As one of the most common human genetic disorders, Down syndrome (DS) is characterized by a mild-to-moderate cognitive disability, which mainly results from genes overexpression on chromosome 21. The expression of miR-99a, a gene harboring on chromosome 21, is increased by 50 folds in DS brain samples. This study aims to investigate the effect of miR-99a overexpression in the hippocampus on mouse behaviors and explore the underlying mechanisms. Lentivirus vectors were delivered into the hippocampus for focal miR-99a overexpression in mice. Then behaviors were observed by an open field, elevated plus maze, rotarod motor test, and Morris water maze. The genes affected by miR-99a were identified by RNA sequencing (RNA-seq) and confirmed by quantitative RT-PCR (qRT-PCR) in samples isolated from the hippocampus injected with lentivirus-GFP-miR-99a or lentivirus-GFP vectors. It was found that the expression of miR-99a with intrahippocampal delivery of lentivirus-GFP-miR-99a resulted in reversal learning impairment in mice although it had no influence on motor function and anxiety. Meanwhile, RNA-seq results showed that 92 genes including mRNAs and microRNAs were significantly regulated by miR-99a, consistent with qRT-PCR consequence. Moreover, dual-luciferase reporter assay showed that miR-99a could directly bind to the 3’-untranslated regions (3’UTR) of target genes (Clic6 and Kcnj13) with an inhibitory effect on their activity. Furthermore, we also found that miR-99a overexpression affected different biological processes by bioinformatic analyses. Our study showed that miR-99a overexpression in the hippocampus leads to cognitive impairment through regulating the expressions of various genes, which reveals a novel function of miR-99a and provides new insights into understanding the pathophysiologic process of DS.

作为最常见的人类遗传疾病之一，唐氏综合征（DS）的特点是轻度至中度认知障碍，主要由21 号染色体上的基因过度表达引起。 miR-99a 的表达是 21 号染色体上的基因，在 DS 脑样本中增加了 50 倍。本研究旨在探讨海马区 miR-99a 过表达对小鼠行为的影响，并探讨其潜在机制。将慢病毒载体递送到海马体中以在小鼠中进行局灶性 miR-99a 过表达。然后通过开放场、高架十字迷宫、旋转运动测试和莫里斯水迷宫观察行为. 受 miR-99a 影响的基因通过 RNA 测序 (RNA-seq) 鉴定，并通过定量 RT-PCR (qRT-PCR) 在从注射了慢病毒-GFP-miR-99a 或慢病毒-GFP 载体的海马体分离的样品中确认。一世发现 miR-99a 的表达与慢病毒-GFP-miR-99a 的海马内递送导致小鼠逆转学习障碍，尽管它对运动功能和焦虑没有影响。同时，RNA-seq 结果显示包括 mRNA 和 microRNA 在内的 92 个基因受到 miR-99a 的显着调控，与 qRT-PCR 结果一致。此外，双荧光素酶报告基因分析表明，miR-99a 可以直接与靶基因（Clic6 和 Kcnj13）的 3'-非翻译区 (3'UTR) 结合，对其活性具有抑制作用。此外，我们还通过生物信息学分析发现 miR-99a 过表达影响不同的生物学过程。我们的研究表明，海马中 miR-99a 的过度表达通过调节各种基因的表达导致认知障碍，